Modalities for the treatment of degenerative diseases of the retina

ABSTRACT

This invention relates to methods for improved cell-based therapies for retinal degeneration and for differentiating human embryonic stem cells and human embryo-derived into retinal pigment epithelium (RPE) cells and other retinal progenitor cells.

This application is a divisional of U.S. application Ser. No.13/477,763, filed May 22, 2012, which is a continuation of U.S.application Ser. No. 12/857,911 filed Aug. 17, 2010 (now U.S. Pat. No.8,268,303) which is a continuation of U.S. application Ser. No.11/041,382, filed Jan. 24, 2005 (now U.S. Pat. No. 7,794,704), whichclaims the benefit of U.S. Provisional Application No. 60/538,964, filedJan. 23, 2004, each of which is hereby incorporated by reference hereinin its entirety.

FIELD OF THE INVENTION

This invention relates generally to methods for improved cell-basedtherapies for retinal degeneration and other visual disorders as well astreatment of Parkinson's disease and for differentiating mammalianembryonic stem cells and mammalian embryo-derived cells into retinalpigment epithelium (RPE) cells and other eye tissue including, but notlimited to) rods, cones, bipolar, corneal, neural, iris epithelium, andprogenitor cells.

BACKGROUND OF THE INVENTION

Many parts of the central nervous system (CNS) exhibit laminarorganization, and neuropathological processes generally involve morethan one of these multiple cellular layers. Diseases of the CNSfrequently include neuronal cell loss, and, because of the absence ofendogenous repopulation, effective recovery of function followingCNS-related disease is either extremely limited or absent. Inparticular, the common retinal condition known as age-related maculardegeneration (AMD) results from the loss of photoreceptors together withthe retinal pigment epithelium (RPE), with additional variableinvolvement of internuncial (“relay”) neurons of the inner nuclear layer(INL). Restoration of moderate-to-high acuity vision, therefore,requires the functional replacement of some or all of the damagedcellular layers.

Anatomically, retinitis pigmentosa (RP), a family of inherited retinaldegenerations, is a continuing decrease in the number of photocreceptorcell nuclei which leads to loss of vision. Although the phenotype issimilar across most forms of RP, the underlying cellular mechanisms arediverse and can result from various mutations in many genes. Mostinvolve mutations that alter the expression ofphotoreceptor-cell-specific genes, with mutations in the rhodopsin geneaccounting for approximately 10% of these. In other forms of thedisease, the regulatory genes of apoptosis are altered (for example, Baxand Pax2). AMD is a clinical diagnosis encompassing a range ofdegenerative conditions that likely differ in etiology at the molecularlevel. All cases of AMD share the feature of photoreceptor cell losswithin the central retina. However, this common endpoint appears to be asecondary consequence of earlier abnormalities at the level of the RPE,neovascularization, and underlying Bruch's membrane. The latter mayrelate to difficulties with photoreceptor membrane turnover, which areas yet poorly understood. Additionally, the retinal pigment epitheliumis one of the most important cell types in the eye, as it is crucial tothe support of the photoreceptor function. It performs several complextasks, including phagocytosis of shed outer segments of rods and cones,vitamin A metabolism, synthesis of mucoploysacharides involved in themetabolite exchange in the subretinal space, transport of metabolites,regulation of angiogenesis, absorption of light, enhancement ofresolution of images, and the regulation of many other functions in theretina through secreted proteins such as proteases and proteaseinhibitors.

An additional feature present in some cases of AMD is the presence ofaberrant blood vessels, which result in a condition known as choroidalneovascularization (CNV). This neovascular (“wet”) form of AMD isparticularly destructive and seems to result from a loss of properregulation of angiogenesis. Breaks in Bruch's membrane as a result ofRPE dysfunction allows new vessels from the choroidal circulation accessto the subretinal space, where they can physically disrupt outer-segmentorganization and cause vascular leakage or hemorrhage leading toadditional photoreceptor loss.

CNV can be targeted by laser treatment. Thus, laser treatment for the“wet” form of AMD is in general use in the United States. There areoften undesirable side effects, however, and therefore patientdissatisfaction with treatment outcome. This is due to the fact thatlaser burns, if they occur, are associated with photoreceptor death andwith absolute, irreparable blindness within the corresponding part ofthe visual field. In addition, laser treatment does not fix theunderlying predisposition towards developing CNV. Indeed, laser burnshave been used as a convenient method for induction of CNV in monkeys(Archer and Gardiner, 1981). Macular laser treatments for CNV are usedmuch more sparingly in other countries such as the U.K. There is nogenerally recognized treatment for the more common “dry” form of AMD, inwhich there is photoreceptor loss overlying irregular patches of RPEatrophy in the macula and associated extracellular material calleddrusen.

Since RPE plays an important role in photoreceptor maintenance, andregulation of angiogenesis, various RPE malfunctions in vivo areassociated with vision-altering ailments, such as retinitis pigmentosa,RPE detachment, displasia, athrophy, retinopathy, macular dystrophy ordegeneration, including age-related macular degeneration, which canresult in photoreceptor damage and blindness. Specifically and inaddition to AMD, the variety of other degenerative conditions affectingthe macula include, but are not limited to, cone dystrophy, cone-roddystrophy, malattia leventinese, Doyne honeycomb dystrophy, Sorsby'sdystrophy, Stargardt disease, pattern/butterfly dystrophies, Bestvitelliform dystrophy, North Carolina dystrophy, central areolarchoroidal dystrophy, angioid streaks, and toxic maculopathies.

General retinal diseases that can secondarily effect the macula includeretinal detachment, pathologic myopia, retinitis pigmentosa, diabeticretinopathy, CMV retinitis, occlusive retinal vascular disease,retinopathy of prematurity (ROP), choroidal rupture, ocularhistoplasmosis syndrome (POHS), toxoplasmosis, and Leber's congenitalamaurosis. None of the above lists is exhaustive.

All of the above conditions involve loss of photoreceptors and,therefore, treatment options are few and insufficient.

Because of its wound healing abilities, RPE has been extensively studiedin application to transplantation therapy. In 2002, one year into thetrial, patients were showing a 30-50% improvement. It has been shown inseveral animal models and in humans (Gouras et al., 2002, Stanga et al.,2002, Binder et al., 2002, Schraermeyer et al., 2001, reviewed by Lundet al., 2001) that RPE transplantation has a good potential of visionrestoration. However, even in an immune-privileged site such as the eye,there is a problem with graft rejection, hindering the progress of thisapproach if allogenic transplantation is used. Although newphotoreceptors (PRCs) have been introduced experimentally bytransplantation, grafted PRCs show a marked reluctance to link up withsurviving neurons of the host retina. Reliance on RPE cells derived fromfetal tissue is another problem, as these cells have shown a very lowproliferative potential. Emory University researchers performed a trialwhere they cultured RPE cells from a human eye donor in vitro andtransplanted them into six patients with advanced Parkinson's Disease.Although a 30-50% decrease in symptoms was found one year aftertransplantation, there is a shortage of eye donors, this is not yet FDAapproved, and there would still exist a need beyond what could be met bydonated eye tissue.

Thus far, therapies using ectopic RPE cells have been shown to behavelike fibroblasts and have been associated with a number of destructiveretinal complications including axonal loss (Villegas-Perez, et al,1998) and proliferative vitreoretinopathy (PVR) with retinal detachment(Cleary and Ryan, 1979). RPE delivered as a loose sheet tends to scrollup. This results in poor effective coverage of photoreceptors as well asa multilayered RPE with incorrect polarity, possibly resulting in cystformation or macular edema.

Delivery of neural retinal grafts to the subretinal (submacular) spaceof the diseased human eye has been described in Kaplan et al. (1997),Humayun et al. (2000), and del Cerro et al. (2000). A serious problemexists in that the neural retinal grafts typically do not functionallyintegrate with the host retina. In addition, the absence of an intactRPE monolayer means that RPE dysfunction or disruption of Bruch'smembrane has not been rectified. Both are fundamental antecedents ofvisual loss.

Thus, there exists no effective means for reconstituting RPE in any ofthe current therapies and there remain deficiencies in each,particularly the essential problem of a functional disconnection betweenthe graft and the host retina. Therefore there exists the need for animproved retinal therapy.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide improved methods forthe derivation of eye cells including, but not limited to, neural cells,including horizontal cells and amacrine cells, retinal cells such asrods and cones, corneal cells, vascular cells, and RPE and RPE-likecells from stem cells and to provide improved methods and therapies forthe treatment of retinal degeneration. In particular, these methodsinvolve the use of RPE and RPE-like cells derived from human embryonicstem cells.

One embodiment of the present invention provides an improved method ofgenerating cells for therapy for retinal degeneration using RPE cells,RPE-like cells, the progenitors of these cells or a combination of twoor three of any of the preceding derived from mammalian embryonic stemcells in order to treat various conditions including but not limited toretinitis pigmentosa and macular degeneration and associated conditions.The cell types which can be produced using this invention include, butare not limited to, RPE, RPE-like cells, and RPE progenitors. Cellswhich may also be produced include iris pigmented epithelial (IPE)cells. Vision associated neural cells including internuncial neurons(e.g. “relay” neurons of the inner nuclear layer (INL)) and amacrinecells (interneurons that interact at the second synaptic level of thevertically direct pathways consisting of thephotoreceptor-bipolar-ganglion cell chain—they are synaptically activein the inner plexiform layer (IPL) and serve to integrate, modulate andinterpose a temporal domain to the visual message presented to theganglion cell) can also be produced using this invention. Additionally,retinal cells, rods, cones, and corneal cells can be produced. In afurther embodiment of the present invention, cells providing thevasculature of the eye can also be produced. The cells of the presentinvention may be transplanted into the subretinal space by usingvitrectomy surgery. Non-limiting examples include the transplantation ofthese cells in a suspension, matrix, or substrate. Animal models ofretinitis pigmentosa that may be treated include rodents (rd mouse,RPE-65 knockout mouse, tubby-like mouse, RCS rat, cats (Abyssinian cat),and dogs (cone degeneration “cd” dog, progressive rod-cone degeneration“prcd” dog, early retinal degeneration “erd” dog, rod-cone dysplasia 1,2 & 3 “rcd1, rcd2 & rcd3” dogs, photoreceptor dysplasia “pd” dog, andBriard “RPE-65” (dog). Evaluation is performed using behavioral tests,fluorescent angiography, histology, or functional testing such asmeasuring the ability of the cells to perform phagocytosis(photoreceptor fragments), vitamin A metabolism, tight junctionsconductivity, or evaluation using electron microscopy. One of the manyadvantages to the methods presented here is the ability to produce andtreat many more patients than it would be possible to treat if one werelimited to using eye donor tissue.

A further embodiment of the present invention provides methods for thespontaneous differentiation of hES cells into cells with numerouscharacteristics of RPE. These RPE preparations are capable of phenotypicchanges in culture and maintaining RPE characteristics through multiplepassages. The present invention also provides for methods ofdifferentiation of established RPE cell lines into alternate neuronallineages, corneal cells, retinal cells as a non-limiting example throughthe use of bFGF or FGF.

Another embodiment of the present invention is a method for thederivation of new RPE lines and progenitor cells from existing and newES cell lines. There can be variations in the properties, such as growthrate, expression of pigment, or de-differentiation andre-differentiation in culture, of RPE-like cells when they are derivedfrom different ES cell lines. There can be certain variations in theirfunctionality and karyotypic stability, so it is desirable to providemethods for the derivation of new RPE lines and new ES cell lines whichwould allow choosing the lines with desired properties that can beclonally selected to produce a pure population of high quality RPE-likecells.

Cells which may also be derived from existing and new ES cell linesinclude iris pigmented epithelial (IPE) cells. In an additionalembodiment, vision associated neural cells including internuncialneurons (e.g. “relay” neurons of the inner nuclear layer (INL)) andamacrine cells can also be produced using this invention. Additionally,retinal cells, rods, cones, and corneal cells can be produced. In afurther embodiment of the present invention, cells providing thevasculature of the eye can also be produced.

Another embodiment of the present invention is a method for thederivation of RPE lines or precursors to RPE cells that have anincreased ability to prevent neovascularization. Such cells can beproduced by aging a somatic cell from a patient such that telomerase isshortened where at least 10% of the normal replicative lifespan of thecell has been passed, then the use of said somatic cell as a nucleartransfer donor cell to create cells that overexpress angiogenesisinhibitors such as Pigment Epithelium Derived Factor (PEDF/EPC-1).Alternatively such cells may be genetically modified with exogenousgenes that inhibit neovascularization.

Another embodiment of the present invention utilized a bank of ES orembryo-derived cells with homozygosity in the HLA region such that saidcells have reduced complexity of their HLA antigens.

Therefore, an additional embodiment of the present invention includesthe characterization of ES-derived RPE-like cells. Although theES-derived pigmented epithelial cells strongly resemble RPE by theirmorphology, behavior and molecular markers, their therapeutic value willdepend on their ability to perform RPE functions and to remainnon-carcinogenic. Therefore, the ES-derived RPE cells are characterizedusing one or more of the following techniques: (i) assessment of theirfunctionality, i.e. phagocytosis of the photoreceptor fragments, vitaminA metabolism, wound healing potential; (ii) evaluation of thepluripotency of RPE-like ES cells derivatives through animal modeltransplantations, (as a non-limiting example this can include SCIDmice); (iii) phenoytping and karyotyping of RPE-like cells; (iv)evaluation of ES cells-derived RPE-like cells and RPE tissue by geneexpression profiling, (v) evaluation of the expression of molecularmarkers of RPE at the protein level, including bestrophin, CRALBP,RPE-65, PEDF. The cells can also be evaluated based on their expressionof transcriptional activators normally required for the eye development,including rx/rax, chx10/vsx-2/alx, ots-1, otx-2, six3/optx, six6/optx2,mitf, pax6/mitf, and pax6/pax2 (Fischer and Reh, 2001, Baumer et al.,2003).

An additional embodiment of the present invention is a method for thecharacterization of ES-derived RPE-like cells using at least one of thetechniques selected from the group consisting of (i) assessment of theES-derived RPE-like cells functionality; (ii) evaluation of thepluripotency of RPE-like ES cell derivatives through animal modeltransplantations; (iii) phenoytping and karyotyping of RPE-like cells;(iv) evaluation of gene expression profiling, (v) evaluation of theexpression of molecular markers of RPE at the protein level; and (vi)the expression of transcriptional activators normally required for theeye development. In a further embodiment these techniques may be usedfor the assessment of multiple hES cell-derived cell types.

Another embodiment of the present invention is a method for thederivation of RPE cells and RPE precursor cells directly from human andnon-human animal morula or blastocyst-staged embryos (EDCs) without thegeneration of ES cell lines.

Embryonic stem cells (ES) can be indefinitely maintained in vitro in anundifferentiated state and yet are capable of differentiating intovirtually any cell type. Thus human embryonic stem (hES) cells areuseful for studies on the differentiation of human cells and can beconsidered as a potential source for transplantation therapies. To date,the differentiation of human and mouse ES cells into numerous cell typeshave been reported (reviewed by Smith, 2001) including cardiomyocytes[Kehat et al. 2001, Mummery et al., 2003 Carpenter et al., 2002],neurons and neural precursors (Reubinoff et al. 2000, Carpenter et al.2001, Schuldiner et al., 2001), adipocytes (Bost et al., 2002, Aubert etal., 1999), hepatocyte-like cells (Rambhatla et al., 2003), hematopoeticcells (Chadwick et al., 2003). oocytes (Hubner et all., 2003),thymocyte-like cells (Lin R Y et al., 2003), pancreatic islet cells(Kahan, 2003), and osteoblasts (Zur Nieden et al., 2003). Anotherembodiment of the present invention is a method of identifying cellssuch as RPE cells, hematopoietic cells, muscle cells, liver cells,pancreatic beta cells, neurons, endothelium, progenitor cells or othercells useful in cell therapy or research, derived from embryos,embryonic stem cell lines, or other embryonic cells with the capacity todifferentiate into useful cell types by comparing the messenger RNAtranscripts of such cells with cells derived in-vivo. This methodfacilitates the identification of cells with a normal phenotype and forderiving cells optimized for cell therapy for research.

The present invention provides for the differentiation of human ES cellsinto a specialized cell in the neuronal lineage, the retinal pigmentepithelium (RPE). RPE is a densely pigmented epithelial monolayerbetween the choroid and neural retina. It serves as a part of a barrierbetween the bloodstream and retina, and it's functions includephagocytosis of shed rod and cone outer segments, absorption of straylight, vitamin A metabolism, regeneration of retinoids, and tissuerepair. (Grierson et al., 1994, Fisher and Reh, 2001, Marmorstein etal., 1998). The RPE is easily recognized by its cobblestone cellularmorphology of black pigmented cells. In addition, there are severalknown markers of the RPE, including cellular retinaldehyde-bindingprotein (CRALBP), a cytoplasmic protein that is also found in apicalmicrovilli (Bunt-Milam and Saari, 1983); RPE65, a cytoplasmic proteininvolved in retinoid metabolism (Ma et al., 2001, Redmond et al., 1998);bestrophin, the product of the Best vitelliform macular dystrophy gene(VMD2, Marmorstein et al., 2000), and pigment epithelium derived factor(PEDF) a 48 kD secreted protein with angiostatic properties (Karakousiset al., 2001, Jablonski et al., 2000).

An unusual feature of the RPE is its apparent plasticity. RPE cells arenormally mitotically quiescent, but can begin to divide in response toinjury or photocoagulation. RPE cells adjacent to the injury flatten andproliferate forming a new monolayer (Zhao et al, 1997). Several studieshave indicated that the RPE monolayer can produce cells of fibroblastappearance that can later revert to their original RPE morphology(Grierson et al., 1994, Kirchhof et al., 1988, Lee et al., 2001). It isunclear whether the dividing cells and pigmented epithelial layer arefrom the same lineage as two populations of RPE cells have beenisolated: epithelial and fusiforms. (McKay and Burke, 1994). In vitro,depending on the combination of growth factors and substratum, RPE canbe maintained as an epithelium or rapidly dedifferentiate and becomeproliferative (Zhao 1997, Opas and Dziak, 1994). Interestingly, theepithelial phenotype can be reestablished in long-term quiescentcultures (Griersion et al., 1994).

In mammalian development, RPE shares the same progenitor with neuralretina, the neuroepithelium of the optic vesicle. Under certainconditions, it has been suggested that RPE can transdifferentiate intoneuronal progenitors (Opas and Dziak, 1994), neurons (Chen et al., 2003,Vinores et al., 1995), and lens epithelium (Eguchi, 1986). One of thefactors which can stimulate the change of RPE into neurons is bFGF (Opazand Dziak, 1994, a process associated with the expression oftranscriptional activators normally required for the eye development,including rx/rax, chx10/vsx-2/alx, ots-1, otx-2, six3/optx, six6/optx2,mitf, and pax6/pax2 (Fischer and Reh, 2001, Baumer et al., 2003).Recently, it has been shown that the margins of the chick retina containneural stem cells (Fischer and Reh, 2000) and that the pigmented cellsin that area, which express pax6/mitf, can form neuronal cells inresponse to FGF (Fisher and Reh, 2001).

The present invention provides for the derivation of trabecular meshworkcells from hES and also for genetically modified trabecular meshworkcells for the treatment of glaucoma.

The present invention also provides for the derivation of trabecularmeshwork cells from RPE progenitors and RPE-like cells and also forgenetically modified trabecular meshwork cells for the treatment ofglaucoma.

The present invention includes methods for the derivation of RPE cellsand RPE precursor cells directly from human and non-human animal morulaor blastocyst-staged embryos (EDCs) without the generation of ES celllines, comprising a) maintaining ES cells in vitro in anundifferentiated state; b) differentiating the ES cells into RPE and RPEprecursor cells; and, c) identifying cells the RPE cells by comparingthe messenger RNA transcripts of such cells with cells derived in-vivo.

Further provided by the present invention are methods for the derivationof RPE lines or precursors to RPE cells that have an increased abilityto prevent neovascularization, said methods comprising: a) aging asomatic cell from an animal such that telomerase is shortened wherein atleast 10% of the normal replicative lifespan of the cell has beenpassed; and, b) using the somatic cell as a nuclear transfer donor cellto create cells that overexpress angiogenesis inhibitors, wherein theangiogenesis inhibitors can be Pigment Epithelium Derived Factor(PEDF/EPC-1).

The present invention provides methods for the treatment of Parkinson'sdisease with hES cell-derived RPE, RPE-like and/or RPE progenitor cells.These may be delivered by stereotaxic intrastriatal implantation with ormicrocarriers. Alternately, they may be delivered without the use ofmicrocarriers. The cells may also be expanded in culture and used in thetreatment of Parkinson's disease by any method known to those skilled inthe art.

Other features and advantages of the invention will be apparent from thefollowing detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-F. is a series of photographs showing the appearance ofpigmented areas (characteristic of RPE cells) in spontaneouslydifferentiating hES cells. FIG. 1A is a photograph of pigmented regionsin a 2.5 month old adherent culture, a well of a E-well plate, scanned;FIG. 1B is a photograph of pigmented regions in a 2.5 month old culturedgrown in EB, at 45× magnification; FIG. 1C is a photograph of apigmented area of an adherent culture; FIG. 1D is a photograph of apigmented region of an EB grown culture; FIG. 1E is a photograph of theboundary between pigmented region and the rest of the culture, ×200;Figure F same as Figure E but at ×400 magnification. Arrows in A and Bpoint to pigmented regions

FIG. 2A-F. is a series of photographs which show the loss and regain ofpigmentation and epithelial morphology in culture. FIG. 2A is aphotograph showing primary EB outgrowth, 1 week; FIG. 2B is a photographshowing the primary culture of cells, isolated by trypsin, 1 week; FIG.2C is a photograph showing epithelial islet surrounded by proliferatingcells; FIG. 2D is a photograph showing the regain of pigmentation andepithelial morphology in 1 month old culture; FIG. 2E is a photographshowing the culture after 3 passages, ×200 magnification; FIG. 2F showsthe same culture as in E, ×400 magnification, Hoffman microscopy. Blackarrows point to pigmented cells, white arrows show outgrowing cells withno pigment.

FIG. 3 Left Panel (A-D) and Right Panel is a series of photographs andone graph—these show markers of RPE in hES cells-derived pigmentedepithelial cells. FIGS. 3A and 3B are photographs showingimmunolocalization of RPE marker, bestrophin and corresponding phasemicroscopy field, ×200 magnification; FIGS. 3C and 3D are photographsshowing CRALBP and corresponding phase contrast microscopy field, ×400magnification. Arrows show the colocalization of bestrophin (A) andCRALBP (C) to pigmented cells (C,D); arrowheads point to the absence ofstaining for these proteins (A,B) in non-pigmented regions (C,D)

FIG. 3, Right Panel shows a photograph and graph of western blot of celllysates (line hES #36) with antibodies to bestrophin (a) and CRALBP (b);c,d—undifferentiated hES cells, c—control to anti-CRALBP antibody,d—control to anti-bestrophin antibody

FIG. 4 shows photographs which demonstrate the expression of markers ofPax6 (FIG. 4A), Pax2 (FIG. 4E) and mitf (FIG. 4B, FIG. 4F) in RPE-likecells in long-term quiescent cultures. FIG. 4C, FIG. 4G—phase contrast,FIG. 4D, FIG. 4H—merged images of Pax6/mitf/phase contrast (FIG. 4A,FIG. 4B, FIG. 4C) and Pax2/mitf/phase contrast (FIG. 4E, FIG. 4F, FIG.4G).

FIG. 5A-B show photographs of RPE differentiation in the culture ofhuman embryo-derived cells: bypassing the stage of derivation of ES celllines.

FIG. 6 shows the transcriptional comparison of RPE preparations. FIG.6A-F—Based on the Ontological annotation, this table represents theexpression patterns of RPE related genes for hES cell-derived retinalpigment epithelium (hES-RPE), hES cell derived transdifferentiated(hES-RPE-TD), ARPE-19 and D407, and freshly isolated human RPE (fe-RPE).FIG. 6G—Further data mining revealed known RPE specific ontologies, suchas melanin biosynthesis, vision, retinol-binding, only in fetal RPE andES-RPE but not ARPE-19.

DETAILED DESCRIPTION OF THE INVENTION

Various embodiments of the invention are described in detail and may befurther illustrated by the provided examples. As used in the descriptionherein and throughout the claims that follow, the meaning of “a,” “an,”and “the” includes plural reference unless the context clearly dictatesotherwise. Also, as used in the description herein, the meaning of “in”includes “in” and “on” unless the context clearly dictates otherwise.

The terms used in this specification generally have their ordinarymeanings in art, within the context of the invention, and in thespecific context where each term is used. Certain terms that are used todescribe the invention are discussed below, or elsewhere in thespecification, to provide additional guidance to the practitioner indescribing the compositions and methods of the invention and how to makeand use them. For convenience, certain terms may be highlighted, forexample using italics and/or quotation marks. The use of highlightinghas no influence on the scope and meaning of a term; the scope andmeaning of a term is the same, in the same context, whether or not it ishighlighted. It will be appreciated that the same thing can be said inmore than one way. Consequently, alternative language and synonyms maybe used for any one or more of the terms discussed herein, nor is anyspecial significance to be placed upon whether or not a term iselaborated or discussed herein. Synonyms for certain terms are provided.A recital of one or more synonyms does not exclude the use of othersynonyms. The use of examples anywhere in this specification, includingexamples of any terms discussed herein, is illustrative only, and in noway limits the scope and scope of the invention so long as data areprocessed, sampled, converted, or the like according to the inventionwithout regard for any particular theory or scheme of action.

Definitions

By “embryo” or “embryonic” is meant a developing cell mass that has notimplanted into the uterine membrane of a maternal host. An “embryoniccell” is a cell isolated from or contained in an embryo. This alsoincludes blastomeres, obtained as early as the two-cell stage, andaggregated blastomeres.

The term “embryonic stem cells” refers to embryo-derived cells. Morespecifically it refers to cells isolated from the inner cell mass ofblastocysts or morulae and that have been serially passaged as celllines.

The term “human embryonic stem cells” (hES cells) refers humanembryo-derived cells. More specifically hES refers to cells isolatedfrom the inner cell mass of human blastocysts or morulae and that havebeen serially passaged as cell lines and can also include blastomeresand aggregated blastomeres.

The term “human embryo-derived cells” (hEDC) refers to morula-derivedcells, blastocyst-derived cells including those of the inner cell mass,embryonic shield; or epiblast, or other totipotent or pluripotent stemcells of the early embryo, including primitive endoderm, ectoderm, andmesoderm and their derivatives, also including blastomeres and cellmasses from aggregated single blastomeres or embryos from varying stagesof development, but excluding human embryonic stem cells that have beenpassaged as cell lines.

Embryonic stem (ES) cells which have the ability to differentiate intovirtually any tissue of a human body can provide a limitless supply ofrejuvenated and histocompatible cells for transplantation therapy, asthe problem of immune rejection can be overcome with nuclear transferand parthenogenetic technology. The recent findings of Hirano et al(2003) have shown that mouse ES cells can produce eye-like structures indifferentiation experiments in vitro. Among those, pigmented epithelialcells were described, resembling retinal pigment epithelium.

Preliminary experiments carried out at Advanced Cell Technology withprimate and human ES cell lines show that a in a specialized culturesystem these cells differentiate into RPE-like cells that can beisolated and passaged. Human and mouse NT, Cyno parthenote ES cellderivatives have multiple features of RPE: these pigmented epithelialcells express four molecular markers of RPE—bestrophin, CRALBP, PEDF,and RPE65; like RPE, their proliferation in culture is accompanied bydedifferentiation—loss of pigment and epithelial morphology, both ofwhich are restored after the cells form a monolayer and becomequiescent. Such RPE-like cells can be easily passaged, frozen andthawed, thus allowing their expansion.

The inventors have further shown that human ES cells also producemultiple eye (vitreous body)-like structures in differentiationexperiments in vitro. Histological analysis of these structures show apattern of cells consistent with early retinal development, includingaggregates of cells similar to rods and cones.

RPE Transplantation

At present, chronic, slow rejection of the RPE allografts preventsscientists from determining the therapeutic efficacy of this RPEtransplantation. Several methods are being considered to overcome thisobstacle. The easiest way is to use systemic immunosuppression, which isassociated with serious side-effects such as cancer and infection. Asecond approach is to transplant the patient's own RPE, i.e. homografts,but this has the drawback of using old, diseased RPE to replace evenmore diseased RPE. Yet, a third approach is to use iris epithelium (IPE)from the same patient but this has the drawback that IPE may not performall the vision related functions of RPE. Ultimately a method will needto be found to eliminate rejection and then scientists can determine thetrue efficacy of RPE transplantation in AMD and ARMD. Nuclear transferand parthenogenesis facilitate histocompatibility of grated RPE cellsand progenitors.

RPE defects in Retinitis Pigmentosa

Retinitis pigmentosa is a hereditary condition in which the visionreceptors are gradually destroyed through abnormal genetic programming.Some forms cause total blindness at relatively young ages, where otherforms demonstrate characteristic “bone spicule” retinal changes withlittle vision destruction. This disease affects some 1.5 million peopleworldwide. Two gene defects that cause autosomal recessive RP have beenfound in genes expressed exclusively in RPE: one is due to an RPEprotein involved in vitamin A metabolism (cis retinaldehyde bindingprotein), a second involves another protein unique to RPE, RPE65. Oncerejection is conquered, both of these forms of RP should be treatableimmediately by RPE transplantation. This treatment was inconceivable afew years ago when RP was a hopelessly untreatable and a poorlyunderstood form of blindness.

New research in RPE transplantation suggests there is promise for thetreatment of retinal degeneration, including macular degeneration. Inaddition, a number of patients with advanced RP have regained someuseful vision following fetal retinal cell transplant. One of thepatients, for instance, improved from barely seeing light to being ableto count fingers held at a distance of about six feet from the patient'sface. In a second case, vision improved to ability to see lettersthrough tunnel vision. The transplants in these studies were performedby injection, introducing the new retinal cells underneath the existingneural retina. Not all of the cells survived since the transplantedfetal cells were allogeneic (i.e. not genetically-matched), althoughthose that did survive formed connections with other neurons and beginto function like the photoreceptors around them. Approximately a yearafter the first eight people received the transplants, four haverecovered some visual function and a fifth shows signs of doing so.

Three newly derived human embryonic stem cell lines are similar inproperties to those described earlier (Thomson et al. 1998, Reibunoff etal., 2000, Richards et al., 2000, Lanzendorf et al., 2001): theymaintain undifferentiated phenotype and express known markers ofundifferentiated hES cells, Oct-4, alkaline phosphatase, SSEA-3, SSEA-4,TRA-I-60, TRA-I-81 through 45 passages in culture or over 130 populationdoublings. All hES cell lines differentiate into derivatives of threegerm layers in EB or long term adherent cultures and in teratomas. Oneof the differentiation derivatives of hES cells is similar to retinalpigment epithelium by the following criteria: morphologically, they havea typical epithelial cobblestone monolayer appearance and contain darkbrown pigment in their cytoplasm, which is known to be present in thehuman body only in melanocytes, keratinocytes, retinal and iris pigmentepithelium (IPE). Melanocytes, however, are non-epithelial cells, andkeratynocytes don't secrete but only accumulate melanin. The set ofRPE-specific proteins—bestrophin, CRALBP, PEDF—present in these cellsindicates that they are likely to be similar to RPE and not IPE. Anothersimilarity is the behavior of isolated pigmented cells in culture, whenlittle or no pigment was seen in proliferating cells but was retained intightly packed epithelial islands or re-expressed in newly establishedcobblestone monolayer after the cells became quiescent. Such behaviorwas described for RPE cells in culture (reviewed by Zhao et al., 1997),and it was previously reported (Vinores et al., 1995) that a neuronalmarker tubulin beta III was specifically localized in dedifferentiatingRPE cells in vitro and not in the cells with the typical RPE morphologysuggesting that it reflects the plasticity of RPE and its ability todedifferentiate to a neural lineage. The inventors have observed thesame pattern of tubulin beta III localization in primary and passagedcultures of RPE and RPE-like cells which can reflect a dedifferentiationof such cells in culture or indicate a separate population of cellscommitted to a neuronal fate, that were originally located next topigmented cells through differentiation of hES cells in long-termcultures and could have been co-isolated with RPE-like cells.

In the growing optic vesicle RPE and the neural retina share the samebipotential neuroepithelial progenitor, and their fate was shown to bedetermined by Pax2, Pax6, and Mitf (Baumer et al., 2003), the latterbeing a target of the first two. Pax6 at earlier stages acts as anactivator of proneural genes and is downregulated in the RPE in furtherdevelopment, remaining in amacrine and ganglion cells in mature retina(reviewed by Ashery-Padan and Gruss, 2001). In goldfish, it is alsofound in mitotically active progenitors of regenerating neurons(Hitchcock et al., 1996). The inventors have found that many of theRPE-like cells expressed mitf and Pax6 in a pattern similar to tubulinbeta III and were found only in non-pigmented cells of non-epithelialmorphology that surround pigmented epithelial islands in long termcultures or in cells with a “partial” RPE phenotype (lightly pigmentedand loosely packed). In proliferating cells in recently passagedcultures all these markers were found nearly in every cell suggestingeither a reversal of RPE-like cells to progenitor stage at the onset ofproliferation or massive proliferation of retinal progenitors.Interestingly, in teratomas where islands of pigmented cells ofepithelial morphology were also found, Pax6 was expressed innon-pigmented cells adjacent to pigmented regions (data not shown).Multiple studies have previously shown dedifferentiation of RPE inculture and their transdifferentiation into cells of neuronal phenotype(Reh and Gretton, 1987, Skaguchi et al., 1997, Vinores et al., 1995,Chen et al., 2003), neuronal, amacrine and photoreceptor cells (Zhao etal., 1995), glia (Skaguchi et al., 1997), neural retina (Galy et al.,2002), and to neuronal progenitors (Opaz and Dziak, 1993). Suchprogenitors can in turn coexist with mature RPE-like cells in culture orappear as a result of dedifferentiation of RPE-like cells. At the sametime, cells of neural retina can transdifferentiate into RPE in vitro(Opas et al., 2001), so alternatively, tubulin beta III and Pax6positive cells could represent a transient stage of suchtransdifferentiation of co-isolated neural cells or neural progenitorsinto RPE-like cells.

Differentiation of hES cells into RPE-like cells happened spontaneouslywhen using methods described in the Examples below, and the inventorsnoticed that pigmented epithelial cells reliably appeared in culturesolder than 6-8 weeks and their number progressed overtime—in 3-5 monthscultures nearly every EB had a large pigmented region. In addition tothe described hES lines, six more newly derived hES lines turned intoRPE-like cells, which suggests that since neural fate is usually chosenby ES cells spontaneously, RPE-like cells can arise by default as anadvanced stage of such pathway. It is also possible that in such longterm cultures, where differentiating hES cells form a multi-layeredenvironment, permissive and/or instructive differentiation signals comefrom extracellular matrix and growth factors produced by differentiatingderivatives of hES cells. The model of differentiation of hES cells intoRPE-like cells could be a useful tool to study how such microenvironmentorchestrates RPE differentiation and transdifferentiation.

RPE plays an important role in photoreceptor maintenance, and variousRPE malfunctions in vivo are associated with a number of vision-alteringailments, such as RPE detachment, displasia, athrophy, retinopathy,retinitis pigmentosa, macular dystrophy or degeneration, includingage-related macular degeneration, which can result in photoreceptordamage and blindness. Because of its wound healing abilities, RPE hasbeen extensively studied in application to transplantation therapy. Ithas been shown in several animal models and in humans (Gouras et al.,2002, Stanga et al., 2002, Binder et al., 2002, Schraermeyer et al.,2001, reviewed by Lund et al., 2001) that RPE transplantation has a goodpotential of vision restoration. Recently another prospective niche forRPE transplantation was proposed and even reached the phase of clinicaltrials: since these cells secrete dopamine, they could be used fortreatment of Parkinson disease (Subramanian, 2001). However, even in animmune-privileged eye, there is a problem of graft rejection, hinderingthe progress of this approach if allogenic transplant is used. The otherproblem is the reliance on fetal tissue, as adult RPE has a very lowproliferative potential.

As a source of immune compatible tissues, hES cells hold a promise fortransplantation therapy, as the problem of immune rejection can beovercome with nuclear transfer technology. The new differentiationderivative of human ES cells, retinal pigment epithelium-like cells andthe reliability and simplicity of such differentiation system may offeran attractive potential supply of RPE cells for transplantation.

EXAMPLES Example 1 Spontaneous Differentiation into Pigmented EpithelialCells in Long Term Cultures

When hES cell cultures are allowed to overgrow on MEF in the absence ofLIF, FGF and Plasmanate, they form a thick multilayer of cells. About 6weeks later, dark islands of cells appear within the larger clusters(FIG. 1). These dark cells are easily seen with the naked eye and lookedlike “freckles” in a plate of cells as shown in FIG. 1A. At highermagnification these islands appear as tightly packed polygonal cells ina cobblestone monolayer, typical of epithelial cells, with brown pigmentin the cytoplasm (FIG. 1C). There are differences in the amount ofpigment in the cells with cells in the central part of the islandshaving the most pigment and those near the edges the least. (FIG. 1,E,F).

When hES cells form embryoid bodies (EB)—pigmented epithelial cellsappear in about 1-2% of EBs in the first 6-8 weeks (FIG. 1B). Over timemore and more EBs develop pigmented cells, and by 3 months nearly everyEB had a pigmented epithelial region (FIG. 1D). Morphology of the cellsin the pigmented regions of EBs was very similar to that of adherentcultures (FIG. 1D).

Example 2 Isolation and Culture of Pigmented Epithelial Cells

The inventors isolated pigmented epithelial cells from both adherent hEScell cultures and from EBs. Pigmented polygonal cells were digested withenzymes (trypsin, and/or collagenase, and/or dispase), and the cellsfrom these pigmented islands were selectively picked with a glasscapillary. Although care was taken to pick only pigmented cells, thepopulation of isolated cells invariably contained some non-pigmentedcells. After plating cells on gelatin or laminin for 1-2 days, the cellswere considered to be primary cultures (P0).

Primary cultures contained islands of pigmented polygonal cells as wellas some single pigmented cells. After 3-4 days in culture, non-pigmentedcells that seemed to have lost epithelial morphology (flatter and cellswith lamellipodia) appeared at the periphery of some islands (FIG. 2).The number of such peripheral cells increased over time, suggesting thatthese cells were proliferating, and after 2 weeks most cells in thenewly formed monolayer contained very little or no pigment. Aftercontinued culture, for another 2-3 weeks, pigmented epithelial cellsbegan to reappear, visibly indistinguishable from those in the originalcultures (FIG. 2).

Example 3 Detection of RPE Markers

The preliminary characterization of these differentiated human cells asRPE is based on their similarity to RPE cultures previously described;principally, their epithelial morphology and possession of pigment.There are three types of pigmented epithelial cells in human body:retinal and iris pigmented epithelium and keratinocytes, but the latterdon't secrete pigment. The epithelial structure and cobblestonemorphology are not shared by other pigmented cells, e.g. melanocytes. Itis also noteworthy that RPE cells have been shown to lose and regaintheir pigment and epithelial morphology when grown in culture (Zhao1997, Opas and Dziak, 1994), and the pigmented cells behaved in asimilar manner, so to test the hypothesis that the ES derived cells maybe RPE, they were stained with antibodies to known markers for RPE:bestrophin and CRALBP. FIG. 4 (left panel) shows membrane localizationof bestrophin (A) and CRALBP (C), both are found in pigmented epithelialislands. Not all of the cells stain with these antibodies and intensityof staining correlated with pigment expression and “tightness” ofcolonies—the borders of each pigmented island where cells were largerand more loosely packed showed lower expression of both proteins.

To further characterize presumably RPE cells, analysis was performed onthe expression of bestrophin, CRALBP by Western blotting. FIG. 4 (rightpanel) shows the bands, corresponding to bestrophin, 68 kD (a), CRALBP,36 kD (b) in cell lysates. All these proteins were found in both primarycultures and subsequent passages.

Another known PRE marker, RPE65, was found in the RPE-like cells byreal-time RT-PCR (FIG. 4, right panel, bottom), the

PEDF ELISA assay showed the presence of PEDF in cell lysates of allpresumed RPE cultures, and Western blot showed a band of approximately48 kD (not shown). Detection of markers of neuronal and retinalprogenitors in RPE-like cultures

FIG. 4 shows localization of PAX-6, Pax2, mitf, and tubulin beta III inrecently passaged and old cultures of hES cells-derived RPE. Inproliferating cultures (day 3 after trypsinization, not shown) whereRPE-like morphology of the proliferating cells is lost, nearly everycell showed the presence of mitf, Pax6, tubulin beta III and nestin (notshown). Pax2 was found only a small subset of cells which appearedmitf-negative, while there was a strong degree of co-localization ofPax6/mitf, mitf/tubulin beta III, and Pax6/tubulin beta III. In 21 daysold quiescent cultures after pigmented epithelial islands werereestablished, groups of PAX-6 and mitf were found mostly innon-pigmented cells of non-epithelial morphology between pigmentedepithelial islands (FIG. 4, A-C). and tubulin beta III had a similarpattern of distribution (not shown). However, there were populations ofmitf-positive and Pax6-negative cells, located close to the periphery ofpigmented islands (FIG. 4, A-C). Pax2 was found only in a very smallsubset of mitf-negative cells (FIG. 4, E-H). No presence of either ofthese proteins was ever detected in the cells of “mature” pigmentedepithelial islands. However, these markers in cells that only had someRPE features were often visible, i.e. either looked epithelial but hadno pigment or in certain single pigmented cells away from pigmentedepithelial islands.

Example 4 Characterization of RPE-Like Cells Derived from hES Cell LinesH9 and ACT J-1 from Cyno-1 ES Cells and Derivation of RPE-Like Cellsfrom Existing hES Cell Lines H1 and H7

An RPE-like cell line is expanded, tested for freezing and recovery, andcharacterized using the following methods and molecular markers of RPEcells: bestrophin and CRALBP by Western blot and immunofluorescence,PEDF by ELISA and Western blot, and REP65 by RT-PCR. The cells areinjected in SCID mice with undifferentiated hES or Cyno-1 cells as acontrol to evaluate tumorigenicity. Karyotyping of RPE-like cells willbe done by a clinical laboratory on a commercial basis. Characterizationof the functional properties of RPE-like cells and studies of theirtransplantation potential are then carried out as otherwise described inthis application and also using those techniques known to those skilledin the art.

Gene expression profiling experiments are done using Affymetrix humangenome arrays. Gene expression is compared in RPE-like cells derivedfrom ES cells and in retinal samples from autopsies. Several animalmodels can be used to verify the effectiveness of the transplantedRPE-like cells, including but not limited to, rhesus monkey, rat, andrabbit.

Example 5 Optimization of the Differentiation Culture System EnsuringHigh Yields of RPE-Like Cells

ES cells are cultured on feeder cells or as embryoid bodies (EB) in thepresence of bFGF, insulin, TGF-beta, IBMX, bmp-2, bmp-4 or theircombinations, including stepwise addition. Alternatively, ES cells aregrown on various extracellular matrix-coated plates (laminin,fibronectin, collagen I, collagen IV, Matrigel, etc.) in evaluating therole of ECM in RPE formation. Expression of molecular markers of earlyRPE progenitors (Pax6, Pax2, mitf) and of RPE cells (CRALBP, bestrophin,PEDF, REP65) are evaluated at various time intervals by real-time RT-PCRto verify and determine successful combinations of the above mentionedagents and stepwise procedure that produces enrichment in RPE-like cellsor their progenitors. This approach can also be used to produce commonprogenitors of RPE and other eye tissues, such as photoreceptor orneural retina which can be isolated and further characterized for theirdifferentiation potential and used in transplantation studies.

Example 6 Derivation of RPE and Other Eye Tissue Progenitors fromExisting and New ES Cell Lines

Using the data from the gene expression profiling, expression of the RPEprogenitor markers will be correlated with the expression of the surfaceproteins in order to find a unique combination of surface markers forRPE progenitor cells. If such markers are found, antibodies to surfaceproteins can be used to isolate a pure population of RPE progenitorsthat can be then cultured and further differentiated in culture or usedin transplantation studies to allow their differentiation aftergrafting.

If the data from the gene expression profiling experiments isinsufficient, to isolate the RPE progenitors the following approach willbe used. ES cells and RPE-like cells will be transfected with GFP underthe control of a Pax6 promoter, and stable transfectants will beselected. From a culture of transfected differentiating ES cells orproliferating (dedifferentiated) RPE cells, GFP/Pax6-positive cells willbe isolated by FACS and used as an antigen source for mouse injection toraise monoclonal antibodies to the surface molecules of Pax6 positivecells. Because Pax6 is present not only in RPE progenitors, screeningwill be done (by FACS) using several strategies: a) againstproliferating RPE-like cells, b) against Pax2-positive RPE cells, c)against mitf-positive RPE cells. For b) and c) RPE cells will betransfected with GFP under the corresponding promoter; as a negativecontrol, RPE or ES cells negative by these antigens will be used. Afterexpansion of positive clones selected by all three strategies,antibodies will be tested against all types of cells used in screeningand further analyzed: since this strategy can produce antibodies thatrecognize cell surface antigens specific and non-specific for RPEprogenitors, the cells from differentiating total population of ES cellsor of RPE cells selected with these antibodies will be assessed formolecular markers of RPE progenitors and for their ability to produceRPE.

Using the optimized defined stepwise procedures to produce RPE or otherearly progenitors of eye tissues and the antibodies to their uniquesurface markers, such progenitors will be isolated from differentiatedES cells and cultured in vitro. Their ability to differentiate intovarious tissues of the eye will be investigated using the strategydescribed in Aim 2.

Three ES cell lines that already produced RPE-like cells (H9, ACT J-1,Cyno-1), RPE-like cells will be used to continue to derive RPE-likecells and their progenitors as described in Aims 1 and 2, and H1 and H7hES cell lines will be used to produce new RPE-like cell lines. Afterexpansion and characterization for molecular markers of RPE, these lineswill be single-cloned, and the resulting lines will be characterized asdescribed in Aim 1. The lines meeting criteria for RPE cells will beused for transplantation studies. New human ES cell lines will bederived from unused IVF embryos, from donated oocytes, stimulated todevelop without fertilization (parthenote), and from generateddeveloping blastocysts obtained from donated oocytes with theapplication of nuclear transfer technology. RPE-like cells and commoneye progenitors will be, derived from these lines using the approach inAim 2, and the resulting lines will be characterized as in Aim 1.[Optional] new human ES cell lines will be derived in a virus-freesystem, characterized and submitted for clinical trials.

Example 7 Therapeutic Potential of RPE-Like Cells and Progenitors inVarious Animal Models of Retinitis Pigmentosa & Macular Degeneration

Primate ES cells are tested in cynomologus monkeys (Macaques).Initially, vitrectomy surgery is performed and the cells aretransplanted into the subretinal space of the animals. The first step isthe transplantation of the cells in the suspension format after which asubstrate or matrix is used to produce a monolayer transplantation. Thiscan also be performed in immunosuppressed rabbits using cells derivedfrom human ES-cells and also in various other animal models of retinitispigmentosa, including rodents (rd mouse, RPE-65 knockout mouse,tubby-like mouse, RCS rat, cats (Abyssinian cat), and dogs (conedegeneration “cd” dog, progressive rod-cone degeneration “prcd” dog,early retinal degeneration “erd” dog, rod-cone dysplasia 1, 2 & 3 “rcd1,rcd2 & rcd3” dogs, Photoreceptor dysplasia “pd” dog, and Briard “RPE-65)dog). Evaluation is performed using fluorescent angiography, histology(whether or not there is photoreceptor restoration and possibly ERG.Functional testing will also be carried out, including phagocytosis(photoreceptor fragments), vitamin A metabolism, tight junctionsconductivity, and electron microscopy.

Example 8 Direct Differentiation of RPE Cells from Human Embryo-DerivedCells

Human blastocyst-staged embryos are plated in the presence of murine orchick embryo fibroblasts with or without immunosurgery to remove thetrophectoderm or directly plates on extracellular matrix protein-coatedtissue cultureware. Instead of culturing and passaging the cells toproduce a human ES cell line, the cells are directly differentiated.

When hEDC cell cultures are allowed to overgrow on MEF in the absence ofLIF, FGF and Plasmanate, they will form a thick multilayer of cells.(Alternate growth factors, media, and FBS can be used to alternatedirect differentiation as is known to those skilled in the art.) About 6weeks later, dark islands of cells will appear within the largerclusters. These dark cells are easily seen with the naked eye and lookedlike “freckles” in a plate of cells as shown in FIG. 5B. At highermagnification these islands appear as tightly packed polygonal cells ina cobblestone monolayer, typical of epithelial cells, with brown pigmentin the cytoplasm (FIG. 5A). There are differences in the amount ofpigment in the cells with cells in the central part of the islandshaving the most pigment and those near the edges the least. (FIG. 5B).

When hEDC cells are directly differentiated they may, though typicallyhave not, formed embryoid bodies (EB). Pigmented epithelial cells appearin about 1-2% of these differentiated cells and/or EBs in the first 6-8weeks. Over time more and more EBs develop pigmented cells, and by 3months nearly every EB had a pigmented epithelial region. Morphology ofthe cells in the pigmented regions of EBs was very similar to that ofadherent cultures.

Materials and Methods:

MEF medium: high glucose DMEM, supplemented with 2 mM GlutaMAX I, and500 u/ml Penicillin, 500 ug/ml streptomycin (all from Invitrogen) and16% FCS (HyCLone). hES Cells Growth medium: knockout high glucose DMEMsupplemented with 500 u/ml Penicillin, 500 ug/mistreptomycin, 1%non-essential amino acids solution, 2 mM GlutaMAX I, 0.1 mMbeta-mercaptoethanol, 4 ng/ml bFGF (Invitrogen), 1-ng/ml human LIF(Chemicon, Temecula, Calif.), 8.4% of Serum Replacement (SR, Invitrogen)and 8.4% Plasmanate (Bayer). Derivation medium contained the samecomponents as growth medium except that it had lower concentration of SRand Plasmanate (4.2% each) and 8.4% FCS and 2× concentration of humanLIF and bFGF, as compared to growth medium. EB medium: same as growthmedium except bFGF, LIF, and Plasmanate; the SR concentration was 13%.RPE medium: 50% EB medium and 50% MEF medium.

hES Cell Lines

The cell lines, hES 35, 36, 45, used for these studies were derived withmodifications of previously reported procedures (Thomson et al., 1998,Reubinoff et al., 2000, Lanzendorf et al., 2001). Human frozenblastocysts (line hES35) or cleaved embryos (lines hES36 and hES45) weredonated to the study, approved by two institutional review board, bycouples who had completed their fertility treatment.

Differentiation experiments were performed with adherent hES cells orwith embryoid bodies (EBs). For adherent differentiation, hES cells wereallowed to overgrow on MEFs until the hES colonies lost their tightborders at which time the culture media was replaced with EB medium(usually, 8-10 days after passaging). The medium was changed every 1-2days. For EB formation, hES cells were trypsinized and cultured in EBmedium on low adherent plates (Costar).

Immunostaining

Cells were fixed with 2% paraformaldehyde, permeabilized with 0.1% NP-40for localization of intracellular antigens, and blocked with 10% goatserum, 10% donkey serum (Jackson Immunoresearch Laboratories, WestGrove, Pa.) in PBS (Invitrogen) for at least one hour. Incubation withprimary antibodies was carried out overnight at 4° C., the secondaryantibodies (Jackson Immunoresearch Laboratories, West Grove, Pa.) wereadded for one hour. Between all incubations specimens were washed with0.1% Tween-20 (Sigma) in PBS 3-5 times, 10-15 minutes each wash.Specimens were mounted using Vectashield with DAPI (Vector Laboratories,Burlingame, Calif.) and observed under fluorescent microscope (Nikon).Localization of alkaline phosphatase was done either by Vector Red(Vector Laboratories, Burlingame, Calif.) to live cells or after thesecond wash during immunostaining according to manufacturer'sinstructions. Antibodies used: bestrophin (Novus Biologicals, Littleton,Colo.), anti-CRALBP antibody was a generous gift from Dr. Saari,University of Washington. Secondary antibodies were from JacksonImmunoresearch Laboratories, and Streptavidin-FITC was purchased fromAmersham.

Isolation and Passaging of RPE-Like Cells

Adherent cultures of hES cells or EBs were rinsed with PBS twice andincubated in 0.25% Trypsin/1 mM EDTA (Invitrogen) at 37° C. until themonolayer loosened. Cells from the pigmented regions were scraped offwith a glass capillary, transferred to MEF medium, centrifuged at 200×g,and plated onto gelatin-coated plates in RPE medium. The medium waschanged after the cells attached (usually in 1-2 days) and every 5-7days after that; the cells were passaged every 2-4 weeks with 0.05%Trypsin/0.53 mM EDTA (Invitrogen).

Western Blot and ELISA

Samples were prepared in Laemmli buffer (Laemmli, 1970), supplementedwith 5% Mercaptoethanol and Protease Inhibitor Cocktail (Roche), boiledfor 5 minutes and loaded onto a 8-16% gradient gel (Bio-Rad, Hercules,Calif.) using a Mini-Protean apparatus; the gels were run at 25-30 mAper gel; proteins were transferred to a 0.2 Nitrocellulose membrane(Schleicher and Shull, Keene, N. H.) at 20 volt overnight. Blots werebriefly stained with Ponceau Red (Sigma) to visualize the bands, washedwith Milli-Q water, and blocked for 1 hour with 5% non-fat dry milk in0.1% TBST (Bio-Rad). Primary antibodies to bestrophin, CRALBP or PEDF(Chemicon) were added for 2 hours followed by three 15-minute washeswith TBST; peroxidase-conjugated secondary antibodies were added for 1hour, and the washes were repeated. Blots were detected using ECL systemwith Super-Signal reagent (Pierce). PEDF ELISA was performed on celllysates using PEDF ELISA kit (Chemicon) according to manufacturer'sprotocol.Real-Time RT-PCR

Total RNA was purified from differentiating ES cultures by a two-stepprocedure Crude RNA was isolated using Trizol reagent (Invitrogen) andfurther purified on RNeazy minicolumns (Qiagen). The levels of RPE65transcripts were monitored by real-time PCR using a commercial primerset for RPE65 detection (Assay on Demand # Hs00165642_m1, AppliedBiosystems) and Quantitect Probe RT-PCR reagents (Qiagen), according tothe manufacturer's (Qiagen) protocol.

Derivation and Characterization of Undifferentiated hES Cell Lines

Two female one male hES cell lines were used in these studies. Detailson the derivation of these hES lines are reported elsewhere. All lineshave been passaged more than 50 times during which time they maintain anundifferentiated colony morphology, high alkaline phosphatase activity,and express Oct-4, SSEA-3, SSEA-4, TRA I-60, and TRA I-81 (data notshown). Two lines have normal karyotype (hES36, hES35), while there wereboth normal and aneuploid subpopulations in hES45. Upon spontaneousdifferentiation both in vitro and in teratomas all lines expressed themarkers of three germ layers—muscle actin, alpha-fetoprotein, andtubulin beta III.

Example 9 Use of Transcript Genomics to Identify Normal DifferentiatedCells Differentiated Ex Vivo

Transcriptomics—hES-cell derivatives are likely to play an importantrole in the future of regenerative medicine. Qualitative assessment ofthese and other stem cell derivatives remains a challenge that could beapproached using functional genomics. We compared the transcriptionalprofile of hES-RPE vs. its in vivo counterpart, fetal RPE cells, whichhave been extensively researched for its transplantation value. Bothprofiles were then compared with previously published (Rogojina et al.,2003) transcriptomics data on human RPE cell lines.

The gene expression profile of our data set was compared to two humanRPE cell lines (non-transformed ARPE-19 and transformed D407, Rogojinaet al., 2003) to determine whether hES-RPE have similar globaltranscriptional profiles. To account for common housekeeping genesexpressed in all cells, we used publicly available Affymetrix data setsfrom undifferentiated hES cells (H1 line, h1-hES,—sato et al., 2003) andbronchial epithelial cells (BE, Wright et al., 2004) as a control basedon its common epithelial origin that would allow to exclude commonhousekeeping and epithelial genes and identify RPE-specific genes.

There were similarities and differences between hES-RPE, hES-RPE-TD,ARPE-19, D407. The similarities were further demonstrated by analyzingthe exclusive intersection between those genes present inhES-RPE/ARPE-19 but not in BE (1026 genes). To account for background,we compared this to the exclusive intersection of genes present inBE/hES-RPE, but not ARPE-19 (186 genes), which results in a five- tosix-fold greater similarity in hES-RPE and ARPE-19 when compared to BE.D407/ARPE19 appear to lose RPE specific genes such as RPE65, Bestrophin,CRALBP, PEDF, which is typical of long-term passaged cells (FIG. 6).Further data mining revealed known RPE specific ontologies such asmelanin biosynthesis, vision, retinol-binding, only in fetal RPE andES-RPE but not ARPE19.

Comparison of hES-RPE, ARPE-19 and D407 to their in vivo counterpart,freshly isolated human fetal RPE (feRPE), was in concordance with ourprevious data, demonstrating that the transcriptional identity ofhES-RPE to human feRPE is significantly greater than D407 to fe RPE (2.3fold difference—849 genes/373 genes) and ARPE-19 to feRPE (1.6 folddifference—588 genes/364 genes (FIG. 5c /5 d). The RPE specific markersidentified above, which were only present in hES-RPE and not in ARPE-19or D407 were also present in feRPE, demonstrating a higher similarity ofhES-RPE to its in vivo counterpart than of the cultured RPE lines.

Seven-hundred-and-eighty-four genes present in hES-RPE were absent infeRPE and ARPE-19 data sets. Since the retention of “sternness” genescould potentially cause transformation of hES derivatives into malignantteratomas if transplanted into patients, we created a conservativepotential “sternness” genes data using currently available Affymetrixmicroarray data sets Abeyta et. al 2004 Sato 2003). This resulted in alist of 3806 genes present in all 12 data sets (including commonhousekeeping genes). j Only 36 of the 784 genes present in the hES-RPEdats et but not feRPE-ARPE-19 were common to the 3806 potentialsternness genes. None of these were known sternness genes such as Oct4,Sox2, TDGF1.

Example 10 Use of RPE Cells for Treatment of Parkinson's Disease

hRPE can be used as an alternative source of cells for cell therapy ofParkinson's Disease because they secrete L-DOPA. Studies have showedthat such cells attached to gelatin-coated microcarriers can besuccessfully transplanted in hemiparkinsonian monkeys and producednotable improvements (10-50) thousand cells per target), and inFDA-approved trial started in 2000 the patients received hRPEintrastriatial transplants without adverse effects. One of the manyadvantages to the use of hES cell-derived RPE is that it circumvents theshortage of donor eye tissue. It also facilitates the use of genetherapy.

Other Embodiments

From the foregoing description, it will be apparent that variations andmodifications may be made to the invention described herein to adopt itto various usages and conditions.

We claim:
 1. A composition comprising isolated human RPE cells obtainedby in vitro differentiation of human pluripotent cells that expressOct-4, alkaline phosphatase, SSEA-3, SSEA-4, TRA-I-60, and TRA-I-81,wherein said human RPE cells express RPE65 and bestrophin and whereinsaid composition is suitable for transplantation or injection into ahuman patient in need thereof.
 2. A composition of claim 1 wherein thehuman RPE cells are present in a monolayer.
 3. A frozen preparation ofhuman RPE cells obtained by in vitro differentiation of humanpluripotent cells that express Oct-4, alkaline phosphatase, SSEA-3,SSEA-4, TRA-I-60, and TRA-I-81, which frozen human RPE cells uponthawing are suitable for use in treating a human patient.
 4. Thecomposition or preparation of claim 1 or 3, wherein said human RPE cellsexpress CRALBP, PEDF, RPE65 and bestrophin.
 5. The composition orpreparation of claim 1 or 3, which comprises human RPE cells thatexhibit a cobblestone, epithelial-like, polygonal appearance.
 6. Thecomposition or preparation of claim 1 or 3, which comprises human RPEcells that express CRALBP, PEDF, RPE65 and bestrophin and exhibit acobblestone, epithelial-like, polygonal appearance.